Enrichment of Terbium(III) under synergistic effect of biosorption and biomineralization by Bacillus sp. DW015 and Sporosarcina pasteurii

ABSTRACT Biosorption and biomineralization are commonly used for the immobilization of metal ions. Biosorption is commonly used as a green method to enrich rare earth ions from wastewater. However, little attention has been paid to the facilitating role of biomineralization in the enrichment of rare earth ions. In this study, a strain of Bacillus sp. DW015, isolated from ion adsorption type rare earth ores and a urease-producing strain Sporosarcina pasteurii were used to enrich rare earth elements (REEs) from an aqueous solution. The results indicate that biomineralization accelerates the enrichment of Terbium(III) compared to biosorption alone. Kinetic analysis suggests that the main mode of action of DW015 was biosorption, following pseudo-second-order kinetics (R2 = 0.998). The biomineralization of DW015 did not significantly contribute to the enrichment of Tb(III), whereas excessive biomineralization of S. pasteurii led to a decrease in the enrichment of Tb(III). A synergistic system of biosorption and biomineralization was established by combining the two bacteria, with the optimal mixed bacteria (S. pasteurii:DW015) ratio being 1:19. This study provides fundamental support for the synergistic effect of biosorption and biomineralization and offers a new reference for future microbial-based enrichment methods. IMPORTANCE A weak microbially induced calcium carbonate precipitation (MICP) promotes the enrichment of Tb(III) by bacteria, while a strong MICP leads to the release of Tb(III). However, existing explanations cannot elucidate these mechanisms. In this study, the morphology of the bioprecipitation and the degree of Tb(III) enrichment were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). The data revealed that MICP could drive stable attachment of Tb(III) onto the cell surface, forming a Tb-CaCO3 mixed solid phase. Excessive rapid rate of calcite generation could disrupt the Tb(III) adsorption equilibrium, leading to the release of Tb(III). Therefore, in order for Tb(III) to be stably embedded in calcite, it is necessary to have a sufficient number of adsorption sites on the bacteria and to regulate the rate of MICP. This study provides theoretical support for the process design of MICP for the enrichment of rare earth ions.

surrounding soil and rivers are infiltrated with residual rare earth ions in the smelting wastewater, resulting in wasted resources and a certain negative impact on the flora, fauna, and body of the environment (7,8).Therefore, green and efficient technologies are urgently needed to meet the requirements of sustainable development of ionic rare earths.
A large number of physical and chemical recovery techniques related to rare earths have been reported both domestically and internationally.Some of the cutting-edge technologies include employing magnesium salt-leaching agents to leach rare earth ions instead of ammonium salt-leaching agents to reduce environmental pollution from ammonia-nitrogen wastewater and high-salinity sodium salt wastewater (5).In addition, modified adsorbents are prepared for the selective recovery of rare earths from rare earth wastewater (9).Zhang et al. synthesized two new recyclable rare earth ion adsorb ents, covalent organic framework-p-phenylenediamine-cyanuric chloride, and covalent organic framework-melamine-cyanuric chloride via a one-step solvothermal method.These adsorbents exhibit adsorption capacities up to 150.88 and 168.19 mg/g at pH 5.5 and t = 35°C, respectively (10).However, these traditional recovery processes are more complicated and cause more rare earth ions loss.Because of the use of large amounts of electrical energy, inorganic leaching agents, organic extractants, and other chemical substances, high energy consumption, secondary pollution caused by the discharge of waste liquids, low recovery rate, and other problems are commonly observed (11).
The cells and spores of Bacillus species have also shown efficiency in the adsorption of rare earth ions due to their special structure with multiple phosphate groups (12,13).Cheng et al. used Bacillus licheniformis to adsorb lanthanum in contaminated water with a maximum adsorption capacity of 113.98 mg/g and enrichment efficiency of up to 97.65% (14).The adsorption process of some microorganisms is usually accompa nied by various biochemical reactions such as biomineralization (15).Lu et al. found that the presence of microbial mineralization doubled the recovery of rare earth ions compared with biosorption alone (16).The formation of minerals is directly or indirectly mediated by interactions between microorganisms and heavy metals, thereby affecting the transport and precipitation of metal ions (17).Biomineralization alters the local environment, creating conditions conducive to adsorption, thus highlighting the unique synergistic effect of biosorption and biomineralization.
Biomineralization is a process of forming complex products of interaction between inorganic mineral ions and organic compound molecules of organisms after undergo ing nucleation, growth, and phase change (17).The more recent application is MICP based on urea hydrolysis, where urea hydrolysis is catalyzed by microbially produced urease to produce carbonate or complex salt crystals.Through this mechanism, metal cations in the environment can be co-precipitated with calcium ions, forming carbonate precipitation or other stable complexes (18)(19)(20).Sporosarcina pasteurii, Sporosarcina aquimarina, and Bacillus megaterium are popular urease-producing bacteria used to induce carbonate precipitation (21,22).It is often applied in fracture repair, geotechnical impermeability sealing, and heavy metal pollution treatment (19,23,24).The calcium carbonate crystals produced by the mineralization reaction increase adsorption sites, encapsulating metal ions on the sites along with mineral coverage (16).Additionally, the utilization of the special surface structure of Bacillus and the alkaline pH conditions generated by growth metabolism fulfill the reaction requirements of MICP (25).However, there are few studies on the recovery of rare earth ions using MICP, the effect of MICP on microbial adsorption is unknown, and the strains that meet the enrichment are scarce.Therefore, it is necessary to screen suitable bacteria, establish and improve the synergistic enrichment system, and further study the enrichment of rare earth ions by biosorption and biomineralization.
In this study, Tb(III), a typical study object of REEs, is selected as the enrichment object.Bacillus strains that are tolerant against high-concentration of rare earth ions and produce urease were isolated.S. pasteurii with high urease activity was used as a control (26,27).The enrichment performance of two bacteria for Tb(III) was studied, and the enrichment process was determined by an adsorption kinetic analysis.A more complete set of biomineralization and adsorption systems was continuously optimized and summarized.Finally, the study characterized the mineralization products and formation processes on the bacterial surface via X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) to elucidate the biosorption and biomineralization mechanisms.

Isolation and identification of urease-producing Bacillus strain
A strain-producing urease named DW015 was obtained from the ionic rare earth mine.The colonies on the plates were smooth and white granules, which could change the urea agar medium from yellow to red.Initial experiments determined its urease activity as a follow-up experimental strain.
The 16S rRNA gene sequencing results of strain DW015 were submitted to NCBI, and a Blast search was conducted to compare them with known nucleic acid sequences.The phylogenetic tree of the strain was constructed by MEGA 11 (Fig. 1).The strain was identified as Bacillus sp.DW015, which is now conserved in the Guangdong Microbial Strain Conservation Center (GDMCC) with the accession number GDMCC No: 62949.

Tolerance of bacterial strains against Tb(III)
The growth curves at different Tb(III) concentrations of DW015 and S. pasteurii were presented in Fig. 2. When the Tb(III) concentration was below 1.26 mmol/L, Tb(III) showed almost no response to the growth of DW015 (Fig. 2a).As the Tb(III) concentration increased, the growth of DW015 was progressively inhibited.The growth was completely stopped at a concentration of 3.78 mmol/L.The semi-lethal concentration of DW015 was 3.09 mmol/L.Tb(III) had a strong inhibitory effect on the growth of S. pasteurii and completely stopped growth at a concentration of 2.52 mmol/L, with a semi-lethal concentration of 1.53 mmol/L (Fig. 2b).These findings suggest that DW015 isolated from the abandoned rare earth mines had stronger adaptability to rare earth ions than S. pasteurii.
Rare earth elements cause toxicity to cells, involving lipid peroxidation along with changes in membrane permeability and polarization (28).Therefore, the toxicity of Tb(III) inevitably causes bacterial death.Dead cells were found to be unable to induce mineralization and exhibited significantly reduced adsorption capacity for metal ions compared to that of living cells (29,30).DW015 had stronger resistance to rare earth ions than S. pasteurii and better maintained its activity to ensure effective adsorption and mineralization during the enrichment of rare earth ions.

Adsorption and mineralization of Tb(III) by Bacillus sp. DW015
The effect of cellular adsorption and adsorption with MICP on the enrichment of Tb(III) from solution was investigated in DW015 with initial concentrations of Tb(III) at 400 µmol/L under the conditions of OD 600 = 1, pH 7.4, adsorption time of 4 h, and temperature of 37°C.When the initial concentration of Tb(III) was 400 µmol/L, the enrichment efficiency via adsorption and MICP increased by 19.1% compared to cellular adsorption from 0 to 1 h (Fig. 3a).The enrichment efficiency of DW015 increased rapidly from 0 to 1 h with increasing contact time, especially at the first contact.The binding sites such as the first exposed groups on the cell surface were utilized instantaneously, leading to a sharp increase in the enrichment efficiency.The enrichment efficiency slowly increased and reached equilibrium after 1 h, with a maximum enrichment efficiency of about 98%.The results showed Bacillus sp.DW015 had a strong adsorption of Tb(III).
The mechanism of MICP for metal ion enrichment has been described in many studies (17,31,32).One explanation suggests that the presence of MICP leads to the coverage of the cell surface with induced calcium carbonate, thereby augmenting the cell-specific surface area to achieve an increase in the Tb(III) adsorption sites (16,33).Consequently, a further increase in the rate of Tb(III) enrichment was achieved.The CO 3 2-of this experiment was obtained mainly through the dissolution of CO 2 in air in an alkaline solution, which was a very slow process (34).Due to the failure to produce significant carbonate precipitation, the exact mechanism for the weak MICP of DW015 to enhance the rate of Tb(III) enrichment was still debatable.Therefore, the mineralization system needs to be further optimized.

Adsorption and mineralization of Tb(III) by S. pasteurii
In order to better explore the effect of MICP on the enrichment of Tb(III), S. pasteurii with high urease activity was applied to treat 400 µmol/L Tb(III).It was clear that Bacillus sp.DW015 had stronger adsorption of Terbium but weaker mineralization than S. pasteurii (Fig. 3a and b). S. pasteurii demonstrated limited Tb(III) adsorption capability, with the enrichment efficiency gradually increasing and stabilizing within 0-2 h, reaching a maximum enrichment efficiency of 59.7% (Fig. 3b).Unlike cellular adsorption, where the enrichment efficiency was only 35.6% at the instant of contact, MICP in S. pasteurii led to a significant enhancement in the Tb(III) enrichment efficiency, reaching 95.4% upon contact initiation.This exhibited a completely different trend from the cellular adsorption curve.The highest enrichment efficiency was reached at the instant of contact, followed by a slow decrease over time.It gradually reached the equilibrium at 1 h and the Tb(III) enrichment efficiency remained at about 80%.When dilute hydrochloric acid was added dropwise to the precipitate excluding the supernatant, the precipitate showed noticeable bubbles.It was thus clear that the enrichment of rare earth ions by S. pasteurii was mainly attributed to MICP within a short period of contact.

Adsorption kinetic model of Tb(III) by Bacillus sp. DW015
From the correlation coefficients (R 2 ) of the fitted results (Table 1) and the fitted graphs (Fig. 3c and d), the fitted results of the adsorption process of Tb(III) by both modes of action of DW015 were poorer than those of the pseudo-first-order kinetic model, with R 2 values of 0.543 and 0.816, respectively.The R 2 values of the pseudo-second-order kinetic model fitted by the kinetic model were all >0.99, and the fitted adsorption amount did not differ much from the actual equilibrium adsorption amount derived from the experiment, indicating that the adsorption of Tb(III) by both modes of action of Bacillus sp.DW015 was more consistent with the pseudo-second-order kinetic model.q t = 1/ (0.0032/t + 0.00292) is the adsorption amount of DW015 adsorption with MICP at t, while the adsorption amount of DW015 cellular adsorption at t is q t = 1/(0.0191/t+ 0.00289).
The adsorption process of biosorbents mostly conformed to the pseudo-secondorder kinetic model, and the two adsorption modes of DW015 aligned more closely with the pseudo-second-order kinetic model, which was consistent with the experimental results (35,36).According to some previous studies, it is known that the adsorption of Tb(III) by DW015 is mainly chemisorption, where the mass transfer resistance occurs in the outer cell layer, and the adsorption depends on the action of various groups on the cell surface, forming chemical bonds stronger than physical adsorption (13,15,37).Under nutrient-deficient conditions, bacteria are unable to provide sufficient CO 3 2and maintain an alkaline pH to induce effective mineralization reactions (25,29).The MICP in the "urea-Ca(II)" mineralization exhibits relatively weak effects on enhancing  the efficiency of Tb(III) enrichment.Therefore, chemisorption on the cell surface plays a dominant role in the enrichment of Tb(III) by DW015.

Optimization of a method for Tb(III) enrichment by Bacillus sp. DW015 and S. pasteurii
In order to enhance the mineralization capacity of the present enrichment system, the study made full use of the strong mineralization capacity of S. pasteurii.The mineralization and adsorption experiments were carried out by adding DW015 and S. pasteurii to the "urea-Ca(II) mineralization system" in a certain ratio (total OD 600 = 1).This solved the low mineralization efficiency of DW015 and ensured the accuracy of the experiments.
When S. pasteurii:DW015 = 1:1 (Fig. 3e), the Tb(III) enrichment efficiency remained stable at about 96% from 0 to 20 min, with an additional stabilization period compared with the mineralization of S. pasteurii alone (Fig. 3b).However, the Tb(III) enrichment efficiency began to decrease with time after 20 min and finally stabilized at 66% after 60 min.The Tb(III) enrichment efficiency decreased by 14% during the equilibrium phase compared to the mineralization of S. pasteurii alone.When S. pasteurii:DW015 = 1:4, the Tb(III) enrichment efficiency stabilized at 98% from 0 to 30 min, and then decreased rapidly after 30 min.When S. pasteurii:DW015 = 1:9, the Tb(III) enrichment efficiency was maintained at 98% from 0 to 60 min and then gradually decreased at a rate significantly smaller than the former.When S. pasteurii:DW015 = 1:19, the Tb(III) enrichment efficiency showed a brief upward trend from 0 to 10 min to reach 97% rapidly and maintain equilibrium.Although Tb(III) enrichment efficiency also showed a decreasing trend after 1 h, the enrichment efficiency remained at a high level from 1 to 6 h above 90%.It is thus clear that a lower proportion of MICP induced by S. pasteurii is sufficient to drive the attachment of Tb(III) on the cell surface, and a higher proportion of DW015 favors stable Tb(III) attachment on the cell surface.The efficient and stable enrichment of Tb(III) can be satisfied by the mixture of S. pasteurii:DW015 = 1:19.
Bacillus, negatively charged various functional groups, mucilage, structures, and groups on the cell surface, can be used as an efficient and sustainable biosorbent for rare earth ions (29,38).When the pH in the environment rises, the ionization balance of the functional groups on the cell surface is altered and deprotonated, increasing its affinity for metal cations and enhancing adsorption (39).Thus, a higher abundance of these functional groups implies a greater affinity for rare earth ions.This suggests that DW015 has more functional groups than S. pasteurii.Additionally, the functional groups have an important contribution to the adsorption of rare earth ions when the pH is adjusted by MICP.
Due to the remarkable mineralization ability of S. pasteurii, the addition of 5% S. pasteurii to DW015 (S. pasteurii:DW015 = 1:19) could compensate for the system deficiency in the difficult MICP reaction.When a rapid increase in the enrichment rate occurred (t = 0-10 min), the Tb(III) enrichment efficiency could reach 97% within 10 min.After a period of equilibrium, the enrichment efficiency slowly decreased but stabilized over 90% within 6 h.However, the adsorption efficiency of DW015 could also reach 98% in the previous experiments.When the bacterial concentration was reduced to half (S. pasteurii:DW015 = 1:19 and total OD 600 = 0.5, Fig. 3f), the cell adsorption efficiency was substantially weakened, with the highest efficiency still not reaching 50%.In contrast, the enrichment efficiency of the "Cell-MICP" remained high, reaching up to 99%.This shows that the promotion of Tb(III) adsorption by cells with MICP is very significant.

XRD analysis of Bacillus before and after mineralization and adsorption
To investigate the chemical properties and precipitation morphology before and after the interaction between bacteria and Tb(III), XRD analysis was performed before and after adsorption and mineralization of Tb(III) by mixed bacteria (S. pasteurii:DW015 = 1:19) (Fig. 4).CaCO 3 was formed gradually with increasing contact time (40).The diffraction peaks observed on the XRD spectra after 2 h of bacterial interaction with Tb(III) using MICP could be well matched with calcite (PDF#47-1743), indicating that the strain-induced mineralization to form calcium carbonate precipitates.Besides, no obvious sharp diffraction peaks were detected on the XRD pattern, indicating that Tb(III) did not form minerals during the cell adsorption and initial period of MICP.Existing studies have shown that only a small portion of the rare earth ions combine with other anions (e.g.oxalate) to precipitate as compounds (18,41,42).The results did not reveal any compounds formed by Tb(III).

SEM-EDS analysis of Bacillus before and after mineralization and adsorption
The precipitates of cell mineralization and adsorption were examined by SEM at two representative stages (t = 10, 120 min).The results of EDS analysis of the elemental signal on the cell surface at each stage are presented in Fig. 5 and 6.Before the interaction with Tb(III), cells were cylindrical in shape, with a smooth surface (Fig. 5a).After 10 min of adsorption with MICP, bright irregular attachments appeared on the cell surface.
The ratio of C, Ca, and Tb increased, indicating that Tb(III) started to be adsorbed while inducing the production of CaCO 3 (Fig. 5b).No minerals were formed on the cell surface before or after the adsorption of cells, with irregular attachments present on their surfaces (Fig. 5c).The proportion of Tb element increased with the increase of adsorption time, which is consistent with the results of existing studies (13,22,26).While the two cases exhibit similarities, there is a large difference in the time required to reach high adsorption rates.Hence, it can be inferred that MICP played an important role in the enrichment process during the period.The surface morphology and the energy spectra of the three corresponding positions of Tb(III) adsorbed by bacteria under the action of MICP were investigated and shown in Fig. 5.When the reaction reached 2 h, MICP induced the production of a large number of squares and a small number of spheres, wrapping the bacteria.As per the XRD results, these square minerals are identified as calcite.The signals of Ca and Tb were greatly enhanced, indicating that the bacteria induced the formation of a large number of  CaCO 3 crystals (Fig. 6a and b).During this period, the cells and the adsorption sites on their surface were almost completely encapsulated.From the Ca signals at the three positions, it can be seen that the proportion of Ca in "b-c-d" was increasing (i.e., the crystalline growth process of CaCO 3 minerals produced by MICP was from amorphous to spherical aragonite and finally to massive calcite) (22,43).The atomic ratio C:O = 1:3 in the lumpy material was identified as CaCO 3 (Fig. 6d).
The proportion of Tb decreased continuously during the growth of CaCO 3 minerals, and a portion of Tb(III) located at the mineralization site was released by Ca(II) exchange.This resulted in a weakened Tb signal, with the vast majority of the Tb signal being masked by the calcium carbonate generated during MICP (16).Therefore, it can be presumed that the vast majority of Tb is encapsulated in calcite after the calcite is formed.This is the reason why the enrichment efficiency of Tb(III) remains high, even when the adsorption sites are masked.It is clear that, from SEM-EDS and XRD results, stronger Tb signals can be detected on the amorphous minerals formed on the cell surface.It further demonstrates similar results to Lu's results (16), that Ca and Tb exist as a Tb-CaCO 3 mixture phase in the initial period of the MICP stage and are gradually encapsulated by the subsequently generated calcite.

Tb(III) enrichment processes of Bacillus with MICP
It has been shown that microorganisms regulate metal ion enrichment and precipitate (44).Microorganisms provide a large number of active sites for adsorption and minerali zation, and MICP reduces the activation energy required for the precipitation of metal ions (29).Intuitively, MICP can drive metal ions [Ca(II) and Tb(III)] toward the nucleation sites, reaching a "supersaturated state" at the cell surface, thus, significantly enhancing the Tb(III) enrichment efficiency.However, as the mineralization reaction proceeds, the nucleation sites become gradually covered by CaCO 3 crystals, and a part of Tb(III) is gradually released into the solution, making the adsorption curve more similar to the release curve.The other part of Tb(III) was encapsulated into CaCO 3 crystals until the formation of stable minerals, at which point no more Tb(III) was released, as evidenced by the curve reaching equilibrium (Fig. 3b).No similar results and reasonable explana tions were found in various literature reports.Therefore, it is presumed that the effect of the MICP in increasing the rate of Tb(III) enrichment occurs mainly in the initial period of reaction or even at the moment of contact.
The mineral growth process of MICP is very similar to the sequestration process of Tb.The overlay of CaCO 3 inevitably displaces the original Tb(III) sites and disrupts the cellular adsorption equilibrium (45).This also explains why the enrichment efficiency of Tb(III) decreased afterward (Fig. 3e).However, as the adsorption capacity of DW015 for Tb(III) is much greater than that of S. pasteurii, the numerous functional groups (carboxyl, hydroxyl, phosphate groups, etc.) on the surface of the bacteria also contribute to the formation of stable calcite.Therefore, Tb(III) will not be easily released but will be encapsulated in the gradually formed mineral (22).It serves a good stabilizing role during the precipitation process.
As shown in Fig. 7, the final putative Tb(III) enrichment processes were (i) Chemisorp tion and MICP sites that were provided by DW015.The CO 3 2-required for MICP was provided by S. pasteurii, increasing the ambient pH and inducing Ca(II) binding to the cell surface; (ii) The "supersaturated state" formed by MICP co-precipitates with Ca to promote rapid attachment of Tb(III) to the surface.At this stage, Tb(III) was precipitated through the combined action of chemisorption and MICP.Free Tb(III) was basically adsorbed completely and CaCO 3 began to form on the cell surface; (iii) As CaCO 3 precipitation continued to increase, vaterite and calcite began to form, and the vast majority of Tb(III) was embedded into CaCO 3 .
This work shows that the high Tb(III) enrichment efficiency (98%) can be achieved by Bacillus sp.DW015 within 30 min, which is mainly attributed to chemisorption.Enhanc ing MICP by supplementation with 5% S. pasteurii can shorten the enrichment time to 10 min or even shorter.Ca(II) and Tb(III) are driven by MICP to nucleation sites on the cell surface, which enhances the enrichment efficiency of Tb(III) during the initial period of crystal production of CaCO 3 .The combined adsorption-mineralization recovery method can be developed as a promising method for enriching rare earth resources.

Isolation of urease-producing microorganisms
The dilution plate method was used to isolate strains from abandoned rare earth mines' soil samples.Strains were inoculated from glycerol tubes onto LB solid medium (10 g peptone, 5 g yeast extract, 10 g NaCl, 20 g agar, and 1,000 mL deionized water) and incubated for 12 h.The activated single colony was selected and inoculated into a urease screening medium and incubated at 37°C for 24 h.Urea agar medium consisted of 2 g potassium dihydrogen phosphate, 5 g sodium chloride, 1 g peptone, 4 mL 0.2% phenol red, 20 g agar, and 890 mL water.It was autoclaved and then cooled to about 50°C.Then 100 mL 20% urea solution and 10 mL 10% glucose solution were filtered and sterilized before being added.
Urea is hydrolyzed by urease-producing bacteria to form NH 4 + and HCO 3 -.An increase in pH in the medium causes the phenol red indicator to turn from yellow to red (32,46).In this way, the eligible strains were screened.

Identification of Bacillus strain based on 16S rRNA gene sequencing
The screened Bacillus strains were cultured in LB liquid and collected when they grew to the logarithmic phase.They were then sent to Sangon Biotech (Shanghai) Co., Ltd. for 16S rRNA gene sequencing.After the products were sequenced, the sequences measured were input to NCBI for comparison, and the phylogenetic tree was constructed using MEGA 11.

Preparation of S. pasteurii culture
S. pasteurii (ATCC11859) was purchased from the Beijing Biological Conservation Center.To activate and recover the strain, CASO medium was prepared with the following composition: 15 g tryptone, 5 g soy peptone, 5 g NaCl, and 900 mL deionized water with pH adjusted to 7.3.It was then autoclaved and cooled to about 50°C before filtering and removing bacteria by adding 100 mL of 20% urea solution.

Analysis of tolerance against Tb(III)
Single colonies of Bacillus were transferred to an LB liquid medium with terbium ion concentrations of 50, 300, 400, 500, 600, and 900 mg/L.They were incubated in a shaker for 12 h at 37°C and with a speed of 180 rpm.The OD 600 of the bacterial solution was measured by hour, and curves were plotted by Origin 2021.
The survival rate of cells was calculated from the bacterial concentrations at equilibrium.The survival rate and the related Tb 3+ concentration were fitted with the logistic function in Origin 2021, with the initial value parameter and final value param eter set to 1 and 0, respectively.The equations of cell survival rate and Tb 3+ concentra tion were derived.The semi-lethal concentration was the Tb 3+ concentration when the survival rate was 50%.

Determination of rare-earth ion Tb(III)
The rare earth ion Tb(III) can be combined with excess 2,6-pyridine dicarboxylic acid (DPA) to form a complex.It fluoresces at a specific wavelength, and its fluorescence intensity is significantly linear with the concentration of rare earth ions in a certain range (12,13).The fluorescence intensity was measured with the excitation wavelength of 275 nm and the emission wavelength of 545 nm by adding 100 µL of 100 mmol/L HEPES buffer and 10 µL of 10 mmol/L DPA solution to 90 µL solution.The results were measured in a 96-well enzyme linked immunosorbent assay (ELISA) plate, and the concentration of rare earth ions in the solution was calculated by Origin 2021.

Adsorption and mineralization of Tb(III) by bacterial strains
The bacterial broth cultured to the logarithmic phase was centrifuged at 4,000 rpm.Following centrifugation, the supernatant was removed, washed twice with sterile water, and finally diluted with sterile saline to obtain OD 600 = 2.The cytosol (OD 600 = 1 after mixing) was mixed with a solution containing Tb(III) with different concentrations (100, 200, 400 µmol/L), 25 mmol/L CaCl 2 , and 2% urea.The volume of the solution was 50 mL and the pH was adjusted to 7 with NaOH and HCl (19,40).They were incubated in a shaker at 37°C and with a speed of 180 rpm.CaCl 2 was chosen as the calcium source for the inhibitory effect of nitrate on urease activity (23).After mineralization, the cytosol loaded with rare earth ions was obtained by centrifugation at 10,000 rpm for 2 min at regular intervals, with Bacillus serving as a control bacterium (40).The experimental groups were set up (as shown in Table 2) (47).Each experiment was repeated three times, and hydrochloric acid was added dropwise to the reacted precipitate to confirm the occurrence of a mineralization reaction in bacteria.The concentration of rare earth ions in the supernatant was measured after centrifugation, and its calculation formula is shown in Equation ( 1): Where R is the enrichment efficiency of rare earth ions, c 0 is the concentration of rare earth ions in the solution before adsorption (µmol/L), and c is the concentration of rare earth ions in the solution after adsorption (µmol/L).
Similar adsorption experiments have been conducted to study the enrichment efficiency by directly inoculating bacteria into the medium containing the target removers (48,49).However, a direct addition of Tb(III) to the medium proved unfeasible.Excessive medium interference with Tb(III) was considered, so the factor of the medium must be excluded to ensure the accuracy of the experiment.

Adsorption kinetic model of Tb(III) by bacterial strains
The relationship between adsorption amount and adsorption time was obtained through adsorption experiments, and the kinetic model was fitted by Origin 2021.The pseudo-first-order Equation and pseudo-second-order Equation are shown in Equations ( 2) and (3), respectively, (2) ln q e − q t = lnq e − K 1 t, (3) t/q t = 1/K 2 q e 2 + t/q e , where q e and q t represent the amount (µmol/L) of Tb(III) adsorbed on bacteria at equilibrium and time t, respectively.K 1 and K 2 are pseudo-first-order and pseudo-sec ond-order adsorption rate constants, respectively.

FIG 2
FIG 2 The growth curve at different Tb(III) concentrations of DW015 (a) and S. pasteurii (b).

FIG 5
FIG 5 SEM images and EDS spectra of mixed bacteria (S. pasteurii:DW015 = 1:19) before and after adsorption and mineraliza tion of Tb(III).P peak covered by Au.Two types of enrichment: cellular adsorption (Cell) and enrichment with MICP (Cell-MICP).

FIG 7
FIG 7 Schematic diagram of Tb(III) enrichment processes by Bacillus sp.DW015 and S. pasteurii.

TABLE 1
Fitting parameters of adsorption kinetic model of DW015 to Tb(III)